Chapter 16 Folding and Pigment Binding of Light-Harvesting Chlorophyll a/b Protein (LHCIIb)

Abstract

The major light-harvesting chlorophyll a/b protein (LHCIIb) is one of the most abundant proteins of the chloroplast in green plants. It contains roughly half of the chlorophylls involved in photosynthesis, and exhibits an unusual ability to self-organize in vitro. Simply mixing the apoprotein, native or recombinant, with as pigments, chlorophyll a, chlorophyll b, and xanthophylls, in detergent solution, suffices to trigger protein folding and the assembly of about 18 pigments in their correct binding sacs. A study of the mechanism of this self-organization seems worthwhile since (1) our knowledge about membrane protein folding is scarce compared to what we know about the folding of water-soluble proteins, (2) the mechanism of LHCIIb formation in vitro may give useful clues about the so-far unknown pathway of its assembly in the chloroplast, and (3) a thorough understanding of the process may facilitate the application of recombinant LHCIIb in hybrid constructs such as photovoltaic devices or the construction of potentially useful proteins or other polymers that spontaneously bind other dyes at a similarly high density. During the assembly of recombinant LHCIIb, the formation of protein secondary structure is triggered by the binding of pigments. Chlorophylls are bound in two apparent kinetic phases. A faster one in the range of tens of seconds reflects the binding of chlorophyll a alone with xanthophylls. During the slower step in the ranee of minutes, mostly chlorophyll h is bound. The intermediate complex lacking chlorophyll h is unstable and susceptible to proteases The resulting two-step model of LHCIIb formation is able to explain why LHCIIb in vivo exhibits an apparently constant chlorophyll a, b ratio although several binding sites have been found to be accessible to both of the two chlorophylls in vitro and in plants over-producing chlorophyll b. Time-resolved electron paramagnetic resonance (EPR) techniques have been established to assess protein folding beyond secondary structure formation during LHCIIb assembly, and first results of EPR-monitored kinetics are shown.